A number of nucleic acid amplification methods, such Polymerase Chain Reaction (PCR), have been developed over the years that generate a single-stranded DNA or RNA product during cycling. This single-stranded product can be potentially detected in real-time or in end-point-type assays using an amplicon-specific complementary oligonucleotide cleavable probe, such as a TaqMan® probe or similar cleavable probe.
TaqMan® probes and similar cleavable probes depend on the 5′-nuclease activity of the DNA polymerase used for PCR to hydrolyze an oligonucleotide that is hybridized to the target amplicon. TaqMan®-type cleavable probes are oligonucleotides that generally include a reporter moiety, such as a fluorescent reporter dye attached to the 5′ end and a quencher moiety coupled to the 3′ end. These probes are designed to hybridize to an internal region of a PCR product. In the unhybridized state, the proximity of the fluorescent tag and the quencher attenuates the generation of a fluorescent signal from the probe. During PCR, when the polymerase replicates a template on which a TaqMan® probe is bound, the 5′-nuclease activity of the polymerase cleaves the probe. This decouples the fluorescent and quenching dyes and quenching no longer occurs. Thus, fluorescence increases in each cycle, proportional to the amount of probe cleavage.
Reporter moieties other than fluorescent-based reporter moieties can also be utilized in nucleic acid amplification techniques and can allow for portable, low-energy consumption microfluidic devices to be utilized. For example, electrochemical reporter moieties, such as ferrocene-based reporter moieties, can be utilized in these techniques. Although electrochemical reporter moieties can offer similar sensitivity and specificity as compared to optical-type reporter moieties, they have generally been more problematic at elevated temperatures (such as those encountered during PCR) due to signal degradation. Additionally, ferrocene-type electrochemical reporter moieties are generally less soluble and can have less chemical stability in the desired oxidation states suitable for use as reporter moieties.
The problems associated to date with the use of electrochemical reporter moieties in nucleic acid amplification and sequencing techniques are obviated by the present disclosure.